Yersinia pestis is designated by the Centers for Disease Control (CDC) and NIAID as a Category A priority bacterial pathogen. Y. pestis is the etiological agent of the plague (Black Death), a transmissible disease that has been responsible for millions of deaths throughout the course of history. Although the natural occurrence of this disease is now relatively rare, the possibility of terrorist groups using Y. pestis as a bioweapon is real. Because of the disease's inherent communicability, rapid clinical course, and high mortality, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management. The long-term goal and commercial application of this application is to develop a plague diagnostic detection kit. The objective of this Phase I research is to generate a luxAB ('light')-tagged phage that can specifically detect Y. pestis. Y. pestis specific phages are currently used by the CDC and the World Health Organization (WHO) as a diagnostic standard for the confirmed identification of Y. pestis;however, the phage lysis assays are laboratory based and require 18-24 h to complete. In contrast, the technology described in this application will not require sample processing, a laboratory environment, or extensive incubation periods. Specific Aim 1 will generate a Y. pestis reporter phage by integrating the bacterial luxAB genes into a non-coding region within the genome of the plague diagnostic phage. Recombinant luxAB-tagged phage will be identified and isolated based on the ability of infected cultures to emit bioluminescence. Specific Aim 2 will perform feasibility studies to demonstrate that the luxAB-phage can effectively be used as a Y. pestis detection system. An attenuated Y. pestis strain (exempt select agent, BSL2 pathogen) will be used. The sensitivity limits of detection, the signal response time, and the dose-response characteristics will be determined. Collectively, this application will generate the proof of principal studies for a novel 'light producing'reporter phage that rapidly and specifically detects Y. pestis. Our preliminary results have demonstrated the feasibility of a similar based approach for the detection of non-encapsulated Bacillus anthracis strains. Since a future direction of NIAID applied research is to "encourage development of multiplex diagnostics" for Category A pathogens (2007 NIAID Strategic Plan for Biodefense Research), one of the long-term goals of Guild Associates is to develop a reporter phage cocktail that can be used for the diagnostic identification of different Category A bacterial pathogens, such as B. anthracis and Y. pestis. PUBLIC HEALTH RELEVANCE: This application is significant because it will generate the proof of principle results for a rapid plague diagnostic test kit. A rapid diagnostic test kit is essential in order to quickly implement public health measures which will save lives and prevent the spread of the disease.